Lassa virus (LASV), the causative agent of Lassa hemorrhagic fever (LHF), causes 2 million infections and 10,000 deaths each year, and further threatens global health security as a potential cause of epidemics and pandemics. Rapid and accurate diagnosis is critical to global health efforts, with a clear effect on LASV treatment, vaccine development, and outbreak containment. As observed in the 2018 Nigeria outbreak, burdensome and time-consuming diagnostic protocols delay results reporting (e.g. 4 days from sample collection), unnecessarily expose healthcare workers to infection, and, by delaying diagnosis in LASV-negative cases, push the healthcare infrastructure beyond its capacity. qPCR-based molecular assays offer the greatest potential for creating rapid and sensitive LASV diagnostic tools, but high genetic diversity has precluded a pan-lineage, universal diagnostic that sensitively and specifically detects all clades of LASV with equal performance. Multiple assays targeting different genomic regions are used in the clinic in an attempt to mitigate viral genetic variability, necessitating time-consuming, sequential diagnostic protocols. Aldatu?s PANDAA technology is a novel platform which enables probe-based qPCR for target detection in highly variable genomic regions by simultaneously adapting and amplifying diverse templates. PANDAA uniquely mitigates the presence of target-proximal polymorphisms to allow otherwise divergent templates to be detected with consensus fluorescent probes with similar sensitivities. Building off of our team?s success in development of PANDAA-based assays for SNPs in HIV, another highly polymorphic pathogen, we propose here to leverage the unique capabilities of PANDAA to mitigate lineage- associated genomic variability and develop a rapid, pan-lineage molecular assay for LASV detection. Preliminary feasibility studies have shown that even our as-yet unoptimized PANDAA reagents detect at least five divergent LASV lineages with near equal sensitivity. In this Phase I proposal, we plan to develop and validate an optimized PANDAA-LASV assay through the following aims: (1) initial design of PANDAA-LASV reagents (primers/probes and buffer) using optimized in-house design workflows; (2) refinement of PANDAA-LASV reagents on divergent genotypes representing all circulating lineages; and (3) analytical and clinical validation of a PANDAA-LASV diagnostic assay prototype with panels to rigorously assess sensitivity and specificity. Successful development and validation of the first pan-lineage PANDAA-LASV assay will precede a clinical diagnostic product that could significantly improve LHF diagnosis, management, and outbreak response, effectively reducing the testing algorithm from two tests to one. This novel, universal detection assay could ultimately be deployed in any endemic region on pre-existing qPCR equipment in central labs, and/or integrated into a closed, point-of-care system with sample processing to radically improve the LHF diagnostic workflow.